Hello, I ran into a serious problem during my experiments. For some reasons, I need glycine at the N-terminus of my ovalbumin (mammalian cell expression, slightly modified protein, ordered from an external company), which is currently obscured by methionine. I thought that it would be cut off during expression by the cells themselves, but for some reason it didn't. I purchased methionine aminopeptidase (from Sigma, https://www.sigmaaldrich.com/PL/pl/product/sigma/m6435?context=product) and tried to cut off the methionine with it, but despite trying it also doesn't work, and still on N- terminus is methionine (confirmed using Mass Spec). What else can I do or what am I doing wrong? Thanks in advance for all the advice.
Methionine aminopeptidase is a metal-dependent enzyme. Did you include a suitable metal ion in your reaction? Mn2+ and Co2+ work well.
If the N-terminus is not surface-exposed, the enzyme may not be able to reach it.
Hello,
If it's possible, you could add a signal peptide between your first methionine and your glycine.
Once at the desired localisation a signal peptidase such as Spase 1 will hydrolyse your signal peptide and your protein will end up with a glycine as its first amino-acid.
Signal peptidase I: cleaving the way to mature proteins.
Auclair SM, et al. Protein Sci. 2012. PMID: 22031009 Free PMC article. Review.
I hope this could help.
Have a good day :-)
Pierre
Hello
Is the enzyme you have is mammalian one, or it is from E.coli, the metal requirement is different based on the source, with the mammalian you need zinc or cobalt. While with that from E. coli you need to include in your reaction either cobalt or iron. So you need to know the source of your enzyme meantime I suggest that you read more about the enzyme you are going to use: its type ( there are 2 types in human), metal requirement, best buffer for its activity assay,.......
Good luck
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